Light-gated integrator for highlighting kinase activity in living cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1138651 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29912142_1.fastq.gz | Fastqsanger.gz | |||
| SRR29912142_2.fastq.gz | Fastqsanger.gz | |||
| SRR29912143_1.fastq.gz | Fastqsanger.gz | |||
| SRR29912143_2.fastq.gz | Fastqsanger.gz | |||
| SRR29912144_1.fastq.gz | Fastqsanger.gz |
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Project description:Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into “permanent” fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.
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Project description:Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into "permanent" fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA signaling heterogeneity in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of GαsR201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.
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Project description:Cellular context is crucial for understanding the complex and dynamic kinase functions in health and disease. Systematic dissection of kinase-mediated cellular processes requires rapid and precise stimulation (“pulse”) of a kinase of interest, as well as global and in-depth characterization (“chase”) of the perturbed proteome under living conditions. We herein developed an optogenetic “pulse-chase” strategy, termed Decaging Kinase coupled proteomics (DeKinomics), for proteome-wide profiling of kinase-driven phosphorylation at second-timescale in living cells. We took advantage of the “gain-of-function” feature of DeKinomics to identify direct kinase substrates and further portrayed the global phosphorylation of understudied receptor tyrosine kinases (RTKs) under native cellular settings. DeKinomics offered a general activation-based strategy to study kinase functions with high specificity and temporal resolution under living conditions.
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Project description:Pausing of RNA polymerase II (RNAPII) in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination through cleavage of nascent RNA and removal of stimulatory phosphorylation. To probe the direct consequences of Integrator activity, we generated a degron system to rapidly deplete the Integrator endonuclease INTS11. Degradation of INTS11 elicits a nearly universal increase in RNAPII escape from promoter regions. However, these RNAPII complexes fail to achieve optimal elongation rates and reveal continued Integrator phosphatase activity. Short transcripts are thus selectively upregulated by INTS11 loss, including many non-coding RNAs, transcription factors and signaling regulators. Together, our data indicate a common function for INTS11 at all RNAPII loci, with differential effects on particular genes, pathways or RNA biotypes reflecting transcript lengths rather than Integrator specificity.
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Project description:Pausing of RNA polymerase II (RNAPII) in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination through cleavage of nascent RNA and removal of stimulatory phosphorylation. To probe the direct consequences of Integrator activity, we generated a degron system to rapidly deplete the Integrator endonuclease INTS11. Degradation of INTS11 elicits a nearly universal increase in RNAPII escape from promoter regions. However, these RNAPII complexes fail to achieve optimal elongation rates and reveal continued Integrator phosphatase activity. Short transcripts are thus selectively upregulated by INTS11 loss, including many non-coding RNAs, transcription factors and signaling regulators. Together, our data indicate a common function for INTS11 at all RNAPII loci, with differential effects on particular genes, pathways or RNA biotypes reflecting transcript lengths rather than Integrator specificity.
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Project description:Pausing of RNA polymerase II (RNAPII) in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination through cleavage of nascent RNA and removal of stimulatory phosphorylation. To probe the direct consequences of Integrator activity, we generated a degron system to rapidly deplete the Integrator endonuclease INTS11. Degradation of INTS11 elicits a nearly universal increase in RNAPII escape from promoter regions. However, these RNAPII complexes fail to achieve optimal elongation rates and reveal continued Integrator phosphatase activity. Short transcripts are thus selectively upregulated by INTS11 loss, including many non-coding RNAs, transcription factors and signaling regulators. Together, our data indicate a common function for INTS11 at all RNAPII loci, with differential effects on particular genes, pathways or RNA biotypes reflecting transcript lengths rather than Integrator specificity.
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Project description:Pausing of RNA polymerase II (RNAPII) in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination through cleavage of nascent RNA and removal of stimulatory phosphorylation. To probe the direct consequences of Integrator activity, we generated a degron system to rapidly deplete the Integrator endonuclease INTS11. Degradation of INTS11 elicits a nearly universal increase in RNAPII escape from promoter regions. However, these RNAPII complexes fail to achieve optimal elongation rates and reveal continued Integrator phosphatase activity. Short transcripts are thus selectively upregulated by INTS11 loss, including many non-coding RNAs, transcription factors and signaling regulators. Together, our data indicate a common function for INTS11 at all RNAPII loci, with differential effects on particular genes, pathways or RNA biotypes reflecting transcript lengths rather than Integrator specificity.
2022-10-28 | GSE200701 | GEO
Project description:Pausing of RNA polymerase II (RNAPII) in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination through cleavage of nascent RNA and removal of stimulatory phosphorylation. To probe the direct consequences of Integrator activity, we generated a degron system to rapidly deplete the Integrator endonuclease INTS11. Degradation of INTS11 elicits a nearly universal increase in RNAPII escape from promoter regions. However, these RNAPII complexes fail to achieve optimal elongation rates and reveal continued Integrator phosphatase activity. Short transcripts are thus selectively upregulated by INTS11 loss, including many non-coding RNAs, transcription factors and signaling regulators. Together, our data indicate a common function for INTS11 at all RNAPII loci, with differential effects on particular genes, pathways or RNA biotypes reflecting transcript lengths rather than Integrator specificity.
2022-10-28 | GSE211732 | GEO
Project description:Pausing of RNA polymerase II (RNAPII) in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination through cleavage of nascent RNA and removal of stimulatory phosphorylation. To probe the direct consequences of Integrator activity, we generated a degron system to rapidly deplete the Integrator endonuclease INTS11. Degradation of INTS11 elicits a nearly universal increase in RNAPII escape from promoter regions. However, these RNAPII complexes fail to achieve optimal elongation rates and reveal continued Integrator phosphatase activity. Short transcripts are thus selectively upregulated by INTS11 loss, including many non-coding RNAs, transcription factors and signaling regulators. Together, our data indicate a common function for INTS11 at all RNAPII loci, with differential effects on particular genes, pathways or RNA biotypes reflecting transcript lengths rather than Integrator specificity.
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Project description:Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration Targeting-Sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3’ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disrupting histone mRNA processing, and leading to polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites. HITseq was used to interrogate chromatin binding sites of of proteins in the complex (including INIP, INTS3, INTS9, INTS11, NABP1, NABP2, NELFA, NELFB, NELFCD, NELFE, SPT5). These proteins were fused to LEDGF-IBD and expressed in mouse LEDGF-/-MEF cells. The fusion proteins then target HIV integration to the chromatin binding sites. The HIV integration sites were identified using linker-mediated PCR and highthroughput sequencing and serve as surrogate for chromatin binding sites. Mapping was done using mouse genome mm9.
2015-01-20 | E-GEOD-65090 | biostudies-arrayexpress